EP3660151B1 - Variant proteases having improved performance and storage stability - Google Patents

Description

The invention is in the field of enzyme technology. The invention relates to proteases whose amino acid sequence has been modified, in particular with regard to use in detergents and cleaning agents, in particular with regard to liquid detergents and cleaning agents, in order to give them better storage stability and/or to improve their cleaning performance, and which nucleic acids coding for them and their production. The invention also relates to the uses of these proteases and methods in which they are used, and agents containing them, in particular washing and cleaning agents, in particular liquid washing and cleaning agents.

Proteases are among the most technically important enzymes of all. They are the longest-established enzymes for detergents and cleaning agents and are contained in practically all modern, high-performance detergents and cleaning agents. They break down proteinaceous soiling on the items to be cleaned. Among these, in turn, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important, which are serine proteases because of the catalytically active amino acids. They act as non-specific endopeptidases and hydrolyze any acid amide bonds that are inside peptides or proteins. Their optimum pH is usually in the clearly alkaline range. The article " Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 . Subtilases are naturally produced by microorganisms. Among these, the subtilisins formed and secreted by Bacillus species should be mentioned in particular as the most important group within the subtilases.

Examples of the subtilisin-type proteases preferably used in detergents and cleaning agents are the subtilisins BPN' and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, and subtilisin DY the enzymes thermitase, proteinase K and the proteases TW3 and TW7, which are to be assigned to the subtilases, but no longer to the subtilisins in the narrower sense, as well as variants of the proteases mentioned which have an amino acid sequence which is altered compared to the starting protease. Proteases are modified in a targeted or random manner using methods known from the prior art and are thus optimized, for example, for use in detergents and cleaning agents. These include point, deletion or insertion mutagenesis or fusion with other proteins or protein parts. Correspondingly optimized variants are known for most of the proteases known from the prior art.

In the international patent applications WO95/23221A1 , WO92/21760A1 , WO2013/060621A1 , WO2015/044206A1 , WO2015/055477A1 and WO2012/080202A1 Variants of the alkaline protease from Bacillus lentus DSM 5483 are disclosed which are suitable for use in detergents or cleaning agents. Furthermore, in the international patent applications WO2011/032988A1 and WO2016/096714A1 as well as the European patent application EP3044302A1 Detergents and cleaning agents disclosed which contain variants of the alkaline protease from Bacillus lentus DSM 5483. The protease variants disclosed in these documents can be changed, in addition to other positions, at positions 3, 4, 99 and/or 199 in the numbering of the alkaline protease from Bacillus lentus DSM 5483 and, for example, at the said positions the amino acids 3T, 4I, 99E or 1991. However, combinations of further changes, as described below, do not emerge from these documents.

In general, only selected proteases are suitable at all for use in liquid surfactant-containing preparations. Many proteases do not show sufficient catalytic performance in such preparations. For the use of proteases in detergents and cleaning agents, therefore, a high catalytic activity under conditions such as those found during a washing cycle and a high storage stability are particularly desirable.

Consequently, liquid formulations containing proteases and surfactants from the prior art have the disadvantage that the proteases contained under standard washing conditions (e.g. in a temperature range of 20° C. to 40° C.) do not have a satisfactory proteolytic activity or are not sufficiently storage-stable , and the formulations therefore do not show optimum cleaning performance on protease-sensitive soiling.

Surprisingly, it has now been found that a protease of the alkaline protease type from Bacillus lentus DSM 5483 or a sufficiently similar protease thereto (based on the sequence identity), which is based on the numbering according to SEQ ID NO: 1 (i) at the positions which corresponding to positions 3, 4, 99 or 199, having amino acid substitutions S3T, V4I, R99E or V199I, and (ii) at least one of the positions corresponding to positions 200, 203, 209 or 212, at least one amino acid substitution, in particular at least one of the amino acid substitutions 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S or 212T, with regard to their storage stability and/or their washing performance and/or their stability compared to the wild-type form (SEQ ID NO:1) or an output variant (SEQ ID NO:2 from WO2013/060621A1 ) is improved and is therefore particularly suitable for use in detergents or cleaning agents.

The invention therefore relates to a protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO:1 whose total length has and based on the numbering according to SEQ ID NO: 1 (i) at the positions corresponding to positions 3, 4, 99 or 199, the amino acid substitutions S3t, V4I, R99E and V199I, and (ii) at least one of the positions corresponding to positions 200, 203, 209 or 212 has at least one amino acid substitution.

Another object of the invention is a method for producing a protease as defined above comprising (i) introducing amino acid substitutions S3T, V4I, R99E and V199I at the positions which, based on the numbering according to SEQ ID NO: 1, positions 3, 4 , 99 or 199, and (ii) introducing at least one amino acid substitution at at least one of the positions that correspond to positions 200, 203, 209 or 212 based on the numbering according to SEQ ID NO: 1, in a starting molecule having an amino acid sequence has at least 70% sequence identity with the amino acid sequence specified in SEQ ID NO: 1 over its entire length.

A protease within the meaning of the present patent application therefore includes both the protease as such and a protease produced using a method according to the invention. All statements on the protease therefore relate both to the protease as such and to the proteases produced by means of corresponding processes.

Further aspects of the invention relate to the nucleic acids coding for these proteases, proteases according to the invention or non-human host cells containing nucleic acids and agents according to the invention comprising proteases, in particular washing and cleaning agents, washing and cleaning methods, and uses of the proteases according to the invention in washing or cleaning agents for removal of proteinaceous soiling.

These and other aspects, features and advantages of the invention will become apparent to those skilled in the art from a study of the following detailed description and claims. Each feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it should be understood that the examples contained herein are intended to describe and illustrate the invention, but not to limit it, and in particular the invention is not limited to these examples.

Numerical ranges given in the format "from x to y" include the stated values. Where multiple preferred numeric ranges are given in this format, it is understood that any ranges resulting from the combination of the different endpoints are also included.

"At least one" as used herein means one or more, ie 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

"Liquid" as used herein includes liquids and gels as well as pasty compositions. It is preferred that the liquid compositions are flowable and pourable at room temperature, but it is also possible for them to have a yield point.

The present invention is based on the inventors' surprising finding that amino acid substitutions at the positions described herein bring about improved storage stability and/or improved cleaning performance of this modified protease in detergents and cleaning agents.

In preferred embodiments, the change(s) according to the invention result(s) (i) at the positions corresponding to positions 3, 4, 99 or 199 based on the numbering according to SEQ ID NO:1, and (ii) at at least one of the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 200, 203, 209 or 212, lead to improved cleaning performance of this modified protease in detergents and cleaning agents on at least one protease-sensitive soiling. Proteases according to the invention consequently enable improved removal of at least one, preferably of several, protease-sensitive stains on textiles and/or hard surfaces, for example dishes.

In preferred embodiments, the change(s) according to the invention result(s) (i) at the positions corresponding to positions 3, 4, 99 or 199 based on the numbering according to SEQ ID NO:1, and (ii) at at least one of the positions Which, based on the numbering according to SEQ ID NO:1, correspond to positions 200, 203, 209 or 212, lead to improved storage stability of this modified protease in detergents and cleaning agents.

In preferred embodiments of the protease according to the invention, the protease has (i) at positions corresponding to positions 3, 4, 99 or 199, the amino acid substitutions S3T, V4I, R99E and V199I and (ii) at least one of the positions corresponding to positions 200, 203, 209 or 212, at least one amino acid substitution selected from the group consisting of 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S and 212T.

In preferred embodiments, the protease according to the invention has (i) at positions corresponding to positions 3, 4, 99 or 199, the amino acid substitutions S3T, V4I, R99E and V199I and (ii) at least one of the positions corresponding to positions 200, 203, 209 or 212 correspond to at least one amino acid substitution selected from the group consisting of 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S and 212T, wherein the combination of the amino acid substitutions from group (i) and the at least one amino acid substitution from group (ii) leads to improved cleaning performance of this modified protease in detergents and cleaning agents on at least one protease-sensitive stain.

In preferred embodiments, the protease according to the invention has (i) at positions corresponding to positions 3, 4, 99 or 199, the amino acid substitutions S3T, V4I, R99E and V199I and (ii) at least one of the positions corresponding to positions 200, 203, 209 or 212 correspond to at least one amino acid substitution selected from the group consisting of 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S and 212T, wherein the combination of the amino acid substitutions from group (i) and the at least one amino acid substitution from group (ii) leads to improved storage stability of this modified protease in detergents and cleaning agents.

In preferred embodiments, the protease according to the invention has (i) at positions corresponding to positions 3, 4, 99 or 199, the amino acid substitutions S3T, V4I, R99E and V199I and (ii) at least one of the positions corresponding to positions 200, 203, 209 or 212 correspond to at least one amino acid substitution selected from the group consisting of 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S and 212T, wherein the combination of the amino acid substitutions from group (i) and the at least one amino acid substitution from group (ii) leads both to improved cleaning performance and to improved storage stability of this modified protease in detergents and cleaning agents.

Certain embodiments of the proteases according to the invention have an improved storage stability. They have increased stability in detergents or cleaning agents compared to the wild-type enzyme (SEQ ID NO: 1) and in particular compared to the starting variant of the protease (SEQ ID NO: 2 from WO2013/060621A1 ), especially when stored for 3 or more days, 4 or more days, 7 or more days, 10 or more days, 12 or more days, 14 or more days, 21 or more days or 28 or more days.

Certain embodiments of the proteases according to the invention can have an increased catalytic activity in detergents or cleaning agents independently of or in addition to the increased storage stability. In various embodiments, the proteases according to the invention can have a proteolytic activity which, based on the wild type (SEQ ID NO: 1) and/or an already performance-enhanced starting variant of the protease (SEQ ID NO: 2) WO2013/060621A1 ) is at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109% or 110%. Such performance-enhanced proteases enable improved washing results on proteolytically sensitive soiling in different temperature ranges, in particular a temperature range from 20°C to 40°C.

Furthermore, preferred embodiments of proteases according to the invention have a particular stability in detergents or cleaning agents, for example with respect to surfactants and/or bleaches and/or chelators, and/or with respect to the effects of temperature, in particular to high temperatures, for example between 50°C and 65°C, in particular 60°C, and/or to acidic or alkaline conditions and/or to pH changes and/or to denaturing or oxidizing agents and/or to proteolytic degradation and/or to a change in redox ratios. With particularly preferred embodiments of the invention, performance-improved and/or more temperature-stable protease variants are consequently provided. With further very particularly preferred embodiments of the invention, performance-enhanced and more temperature-stable protease variants are provided. Such advantageous embodiments of proteases according to the invention consequently enable improved washing results on protease-sensitive stains in a wide temperature range.

In the context of the invention, cleaning performance is understood as meaning the lightening performance on one or more soilings, in particular on laundry or dishes. Within the scope of the invention, both the washing or cleaning agent which comprises the protease or the washing or cleaning liquor formed by this agent and the protease itself have a respective cleaning performance. The cleaning performance of the enzyme thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent. The cleaning performance is preferably determined as indicated below.

Washing liquor is understood to mean the use solution containing the washing or cleaning agent which acts on textiles or fabrics or hard surfaces and thus comes into contact with the soiling present on textiles or fabrics or hard surfaces. The washing liquor is usually formed when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, for example in a dishwasher, a washing machine or in another suitable container.

The proteases according to the invention have enzymatic activity, i.e. they are capable of hydrolyzing peptides and proteins, in particular in a washing or cleaning agent. A protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide/peptide bonds in protein/peptide substrates and is thereby able to cleave proteins or peptides. Furthermore, a protease according to the invention is preferably a mature protease, i.e. the catalytically active molecule without signal and/or propeptide(s). Unless otherwise stated, the sequences given also relate to mature (processed) enzymes in each case.

In various embodiments of the invention, the protease is a free enzyme. This means that the protease can act directly with all components of an agent and, if the agent is a liquid agent, that the protease is in direct contact with the agent's solvent (e.g. water). In other embodiments, an agent may contain proteases that form an interaction complex with other molecules or that form an "envelope" contain. A single or several protease molecules can be separated from the other components of the agent by a structure surrounding them. Such a separating structure can arise from, but is not limited to, vesicles, such as a micelle or a liposome. However, the surrounding structure can also be a virus particle, a bacterial cell or a eukaryotic cell. In various embodiments, an agent may contain Bacillus pumilus or Bacillus subtilus cells expressing the proteases of the invention, or cell culture supernatants of such cells.

In particularly preferred embodiments of the invention, the protease comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO: 1 over its entire length by at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91 .5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5 %, 98%, 98.5%, 98.8%, 99% and 99.5% is identical, and based on the numbering according to SEQ ID NO: 1 (i) has the amino acid substitutions S3T, V4I, R99E and V199I, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212, wherein the at least one amino acid substitution is preferably selected from the group consisting of 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S and 212T is selected.

In the context of the present invention, the feature that a protease has at least one of the specified amino acid substitutions means that it contains one (of the specified) amino acid substitution(s) at the respective position, i.e. at least the specified positions are not otherwise mutated or, for example by fragmentation the protease, are deleted.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm, which is established and commonly used in the prior art (cf. eg Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol. 215:403-410 , and Altschul et al. (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402 ) and is done in principle by the fact that similar sequences of nucleotides or amino acids in the Nucleic acid or amino acid sequences are assigned to each other. A tabular assignment of the relevant positions is referred to as an alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are created with computer programs. For example, the Clustal series (cf. eg Chenna et al. (2003) "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Res. 31:3497-3500 ), T-Coffee (cf. For example, Notredame et al. (2000) "T-Coffee: A novel method for multiple sequence alignments", J. Mol. Biol. 302:205-217 ) or programs based on these programs or algorithms. Sequence comparisons (alignments) are also possible using the Vector ^NTI® Suite 10.3 computer program (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW. Unless otherwise indicated, sequence identity given herein is determined using the BLAST algorithm.

Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is usually given as a percentage of identity, ie the proportion of identical nucleotides or amino acid residues in the same positions or in positions that correspond to one another in an alignment. In the case of amino acid sequences, the broader concept of homology includes conserved amino acid exchanges, i.e. amino acids with similar chemical activity, since these usually have similar chemical activities within the protein. Therefore, the similarity of the compared sequences can also be specified as percent homology or percent similarity. Identity and/or homology statements can be made about entire polypeptides or genes or only about individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by similarities in the sequences. Such areas often have identical functions. They can be small, containing only a few nucleotides or amino acids. Such small regions often perform essential functions for the overall activity of the protein. It can therefore make sense to relate sequence matches only to individual, possibly small areas. However, unless otherwise stated, identity or homology statements in the present application relate to the total length of the particular nucleic acid or amino acid sequence stated.

In the context of the present invention, the indication that an amino acid position corresponds to a numerically indicated position in SEQ ID NO:1 therefore means that the corresponding position is assigned to the numerically indicated position in SEQ ID NO:1 in an alignment as defined above.

In a further embodiment of the invention, the protease is characterized in that its cleaning performance (after storage, for example over 3 weeks) compared to the wild-type enzyme (SEQ ID NO: 1) or a starting variant (SEQ ID NO: 2 from WO2013/060621A1 ) not significant is reduced, ie at least 80% of the reference wash performance, preferably at least 100%, more preferably at least 110% or more.

The cleaning performance can be determined in a washing system that contains a detergent in a dosage between 4.5 and 7.0 grams per liter of wash liquor and the protease, with the proteases to be compared being used in the same concentration (based on active protein) and the cleaning performance opposite of soiling on cotton is determined by measuring the degree of cleaning of the washed textiles. For example, the washing process can take place for 60 minutes at a temperature of 40° C. and the water can have a water hardness between 15.5 and 16.5° (German hardness). The concentration of the protease in the detergent intended for this washing system is 0.001 to 0.1% by weight, preferably 0.01 to 0.06% by weight, based on active, purified protein.

A liquid reference detergent for such a washing system can be composed, for example, as follows (all data in percent by weight): 4.4% alkylbenzenesulfonic acid, 5.6% other anionic surfactants, 2.4% C ₁₂ -C ₁₈ Na salts of Fatty acids (soaps), 4.4% non-ionic surfactants, 0.2% phosphonates, 1.4% citric acid, 0.95% NaOH, 0.01% defoamers, 2% glycerin, 0.08% preservatives, 1% ethanol, remainder demineralized water. The dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9 or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH value range between pH 7 and pH 10.5, preferably between pH 7.5 and pH 8.5.

In the context of the invention, the cleaning performance is determined, for example, at 20° C. or 40° C. using a liquid detergent such as that specified above, the washing process preferably taking place for 60 minutes at 600 rpm.

The degree of whiteness, i.e. the lightening of the soiling, as a measure of the cleaning performance, is determined using optical measuring methods, preferably photometrically. A suitable device for this is, for example, the Minolta CM508d spectrometer. The devices used for the measurement are usually calibrated beforehand with a white standard, preferably a supplied white standard.

A liquid reference hand dishwashing detergent for such a washing system can be composed, for example, as follows (all data in % by weight): 8-20% alkylbenzenesulfonic acid, 30-80% demineralized water, 5.4% NaOH (50%), 7.14 % fatty alcohol ether sulfate, 2.0% NaCl (20%), 0.383% phosphoric acid (H ₃ PO ₄ ; 34%/85%), 0.1% preservative, 0.25% perfume, 1.0% dye, 0.04 % bitterness.

By using the respective protease with the same activity, it is ensured that even if there is a discrepancy in the ratio of active substance to total protein (the values of the specific activity) the respective enzymatic properties, for example the cleaning performance on certain types of soiling, are compared. In general, a low specific activity can be compensated for by adding a larger amount of protein.

Otherwise, methods for determining the protease activity are familiar to the person skilled in the field of enzyme technology and are routinely used by him. For example, such methods are disclosed in Surfactants, Vol. 7 (1970), pp. 125-132 . Alternatively, the protease activity can be determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleaves the substrate and releases pNA. The release of the pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (cf. Del Mar et al., 1979). The measurement is carried out at a temperature of 25°C, at pH 8.6 and at a wavelength of 410 nm. The measurement time is 5 minutes and the measurement interval is 20s to 60s. The protease activity is usually given in protease units (PU). Suitable protease activities are, for example, 2.25, 5 or 10 PU per ml wash liquor. However, the protease activity is not zero.

An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measurement method, preferably a photometric method. The test suitable for this involves the protease-dependent cleavage of the substrate protein casein. This is split by the protease into a large number of smaller partial products. All of these partial products have an increased absorption at 290 nm compared to uncleaved casein, with this increased absorption being determined using a photometer and thus a conclusion being drawn as to the enzymatic activity of the protease.

The protein concentration can be determined using known methods, for example the BCA method (bicinchoninic acid; 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method ( Gornall et al., J. Biol. Chem. 177 (1948): 751-766 ) are determined. In this regard, the active protein concentration can be determined by titrating the active centers using a suitable irreversible inhibitor and determining the residual activity (cf. Bender et al., J.Am. Chem. Soc. 88 , 24 (1966): 5890-5913 ) take place.

In addition to the amino acid changes explained above, proteases according to the invention can have further amino acid changes, in particular amino acid substitutions, -insertions or -deletions. Such proteases are further developed, for example, by targeted genetic modification, ie by mutagenesis methods, and are optimized for specific purposes or with regard to specific properties (for example with regard to their catalytic activity, stability, etc.). Furthermore, nucleic acids according to the invention can be introduced into recombination batches and thus used to generate completely new types of proteases or other polypeptides.

The aim is to introduce targeted mutations such as substitutions, insertions or deletions into the known molecules in order, for example, to improve the cleaning performance of enzymes according to the invention. For this purpose, in particular the surface charges and/or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed. For example, the net charge of the enzymes can be changed in order to influence substrate binding, in particular for use in detergents and cleaning agents. Alternatively or additionally, the stability or catalytic activity of the protease can be increased by one or more corresponding mutations, thereby improving its cleaning performance. Advantageous properties of individual mutations, e.g. individual substitutions, can complement each other. A protease that has already been optimized with regard to certain properties, for example with regard to their stability during storage, can therefore be further developed within the scope of the invention.

The following convention is used here for the description of substitutions that affect exactly one amino acid position (amino acid exchanges): first the naturally occurring amino acid is designated in the form of the internationally customary one-letter code, followed by the associated sequence position and finally the inserted amino acid. Multiple substitutions within the same polypeptide chain are separated by slashes. In the case of insertions, additional amino acids are named after the sequence position. For deletions, the missing amino acid is replaced by a symbol, such as an asterisk or a dash, or a Δ is given in front of the corresponding position. For example, A95G describes the substitution of alanine at position 95 by glycine, A95AG the insertion of glycine after the amino acid alanine at position 95 and A95* or ΔA59 the deletion of alanine at position 95. This nomenclature is known to those skilled in the field of enzyme technology.

A further object of the invention is therefore a protease, which is characterized in that it can be obtained from a protease as described above as a starting molecule by one or more conservative amino acid substitutions, the protease in the count according to SEQ ID NO: 1 at least one of having amino acid substitutions described above. The term "conservative amino acid substitution" means the exchange (substitution) of one amino acid residue for another amino acid residue, which exchange does not result in a change in polarity or charge at the position of the exchanged amino acid, e.g. the exchange of a non-polar amino acid residue for another non-polar amino acid residue. Conservative amino acid substitutions within the scope of the invention include, for example: G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, S=T, G=A=I=V =L=M=Y=F=W=P=S=T.

Alternatively or additionally, the protease is characterized in that it can be obtained as a starting molecule from a protease according to the invention by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which has a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids matches the starting molecule, the protease having (i) amino acid substitutions S3T, V4I, R99E and V199I at positions corresponding to positions 3, 4, 99 or 199, and (ii) at least one amino acid substitution at at least one of positions corresponding to positions 200, 203, 209 or 212.

For example, it is possible to delete individual amino acids at the termini or in the loops of the enzyme without the proteolytic activity being lost or reduced as a result. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can also, for example, also reduce the allergenicity of the enzymes in question and thus improve their usability overall. Advantageously, the enzymes retain their proteolytic activity even after mutagenesis, i.e. their proteolytic activity corresponds at least to that of the starting enzyme, i.e. in a preferred embodiment the proteolytic activity is at least 80%, preferably at least 90%, of the activity of the starting enzyme. Other substitutions can also show advantageous effects. Both individual and several contiguous amino acids can be exchanged for other amino acids.

The amino acid positions are defined here by aligning the amino acid sequence of a protease according to the invention with the amino acid sequence of the protease from Bacillus lentus, as specified in SEQ ID NO:1. Furthermore, the assignment of the positions depends on the mature (mature) protein. This assignment is also to be used in particular when the amino acid sequence of a protease according to the invention comprises a higher or lower number of amino acid residues than the protease from Bacillus lentus according to SEQ ID NO:1. Starting from the positions mentioned in the amino acid sequence of the protease from Bacillus lentus , the positions of change in a protease according to the invention are those which are assigned precisely to these positions in an alignment.

Advantageous positions for sequence changes, in particular substitutions, of the protease from Bacillus lentus, which are preferably transferred to homologous positions of the proteases according to the invention and give the protease advantageous functional properties, are therefore the positions that correspond to the positions described herein in an alignment, ie in the count according to SEQ ID NO:1. In the wild-type molecule of the protease from Bacillus lentus (SEQ ID NO:1), the following amino acid residues are located at the positions mentioned: Q200, Y203, A209, N212.

A further confirmation of the correct assignment of the amino acids to be changed, ie in particular their functional correspondence, can be provided by comparative experiments, according to which the two positions assigned to one another on the basis of an alignment are changed in the two compared proteases in the same way and it is observed whether both the enzymatic activity is altered in the same way. For example, enter Amino acid exchange in a specific position of the protease from Bacillus lentus according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example with an increase in the K _M value, and a corresponding change in the enzymatic parameter, for example also an increase in the K _M value, observed in a protease variant according to the invention, whose amino acid replacement was achieved by the same introduced amino acid, this is to be seen as confirmation of the correct assignment.

1. (a) Einbringen einer ein- oder mehrfachen konservativen Aminosäuresubstitution in die Protease, wobei die Protease aufweist:
   1. i) an den Positionen, die den Positionen 3, 4, 99 oder 199 entsprechen, Aminosäuresubstitutionen S3T, V4I, R99E und V1991, und
   2. ii) an mindestens einer der Positionen, die den Positionen 200, 203, 209 oder 212 entsprechen, mindestens eine Aminosäuresubstitution;
2. (b) Veränderung der Aminosäuresequenz durch Fragmentierung, Deletions-, Insertions- oder Substitutionsmutagenese derart, dass die Protease eine Aminosäuresequenz umfasst, die über eine Länge von mindestens 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 oder 269 zusammenhängenden Aminosäuren mit dem Ausgangsmolekül übereinstimmt, wobei die Protease aufweist:
   1. i) an den Positionen, die den Positionen 3, 4, 99 oder 199 entsprechen, Aminosäuresubstitutionen S3T, V4I, R99E und V1991, und
   2. ii) an mindestens einer der Positionen, die den Positionen 200, 203, 209 oder 212 entsprechen, mindestens eine Aminosäuresubstitution.

All the facts mentioned are also applicable to the methods according to the invention for the production of a protease. Accordingly, a method according to the invention also comprises one or more of the following method steps:

1. (a) introducing into the protease a single or multiple conservative amino acid substitution, wherein the protease has:
   1. i) at positions corresponding to positions 3, 4, 99 or 199, amino acid substitutions S3T, V4I, R99E and V1991, and
   2. ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212;
2. (b) Alteration of the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence which is at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262 , 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids matches the starting molecule, the protease having:
   1. i) at positions corresponding to positions 3, 4, 99 or 199, amino acid substitutions S3T, V4I, R99E and V1991, and
   2. ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212.

All embodiments also apply to the methods according to the invention.

In further embodiments of the invention, the protease or the protease produced using a method according to the invention is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92 .5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5 %, 98.8%, 99% or 99.5% identical to the amino acid sequence given in SEQ ID NO:1 over its entire length. The protease or the protease produced using a method according to the invention has (i) the amino acid substitutions S3T, V4I, R99E and V199I at positions which correspond to positions 3, 4, 99 or 199, and (ii) at least one of the amino acid substitutions 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 212S and 212T in at least one of the positions corresponding to positions 200, 203, 209 or 212, each based on the numbering according to SEQ ID NO:1 . Examples of this are the following Amino acid substitution variants: (i) S3T + V4I + R99E + V199I + Q200L + Y203W; (ii) S3T + V4I + R99E + V199I + N212S; (iii) S3T + V4I + R99E + V199I + Q200L + Y203W + S154D + L256E; (iv) S3T + V4) + R99E + V199I + N74D + Q200L + Y203W; (v) S3T + V4I + R99E + V199I + N74D + S154D + Q200L + Y203W + L256E; (vi) S3T + V4I + R99E + V199I + N74D + N212S; (vii) S3T + V4I + R99E + V199I + N74D + S154D + Y203W + L256E; (viii) S3T + V4I + R99E + V199I + N74D + Y203W; (ix) S3T + V4I + R99E + V199I + N74D + S154D + Q200L + L256E; (x) S3T + V4I + R99E + V199I + N74D + Q200L; (xi) S3T + V4I + R99E + V199I + S154D + Q200L + Y203W; (xii) S3T + V4I + R99E + V199I + Q200L + Y203W + L256E; (xiii) S3T + V4I + R99E + V199I + A136Q + R143W + Y161T + Q200L; (xiv) S3T + V4I + R99E + V199I + N74D + R143Y + A209W + N212S + L256E; (xv) S3T + V4I + R99E + V199I + A136Q + S154D + V171L + Q200L, each based on the numbering according to SEQ ID NO:1, and the variants described in the examples.

Another object of the invention is a protease as described above, which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer. An increase in stability during storage and/or during use, for example during the washing process, means that the enzymatic activity lasts longer and the cleaning performance is therefore improved. In principle, all stabilization options described and/or expedient in the prior art can be considered. Such stabilizations are preferred which are achieved via mutations of the enzyme itself, since such stabilizations do not require any further work steps after the enzyme has been obtained. Examples of sequence changes suitable for this are mentioned above. Other suitable sequence changes are known in the art.

• Veränderung der Bindung von Metallionen, insbesondere der Calcium-Bindungsstellen, beispielsweise durch Austauschen von einer oder mehreren der an der Calcium-Bindung beteiligten Aminosäure(n) gegen eine oder mehrere negativ geladene Aminosäuren und/oder durch Einführen von Sequenzveränderungen in mindestens einer der Folgen der beiden Aminosäuren Arginin/Glycin;
• Schutz gegen den Einfluss von denaturierenden Agentien wie Tensiden durch Mutationen, die eine Veränderung der Aminosäuresequenz auf oder an der Oberfläche des Proteins bewirken;
• Austausch von Aminosäuren, die nahe dem N-Terminus liegen, gegen solche, die vermutlich über nicht-kovalente Wechselwirkungen mit dem Rest des Moleküls in Kontakt treten und somit einen Beitrag zur Aufrechterhaltung der globulären Struktur leisten.

Other stabilization options include:

• Changing the binding of metal ions, in particular the calcium binding sites, for example by exchanging one or more of the amino acid(s) involved in the calcium binding for one or more negatively charged amino acids and/or by introducing sequence changes in at least one of the sequences of both amino acids arginine/glycine;
• Protection against the influence of denaturing agents such as surfactants by mutations that cause a change in the amino acid sequence on or at the surface of the protein;
• Replacement of amino acids that are close to the N-terminus with those that presumably make contact with the rest of the molecule via non-covalent interactions and thus contribute to the maintenance of the globular structure.

Preferred embodiments are those in which the enzyme is stabilized in multiple ways, since multiple stabilizing mutations act additively or synergistically.

Another object of the invention is a protease as described above, which is characterized in that it has at least one chemical modification. A protease with such a change is referred to as a derivative, i.e. the protease is derivatized.

In the context of the present application, derivatives are therefore understood to mean proteins whose pure amino acid chain has been chemically modified. Such derivatizations can, for example, be carried out in vivo by the host cell expressing the protein. In this regard, couplings of low-molecular compounds such as lipids or oligosaccharides are particularly noteworthy. However, derivatizations can also be carried out in vitro , for example by chemically converting a side chain of an amino acid or by covalently binding another compound to the protein. For example, it is possible to couple amines to carboxyl groups of an enzyme to change the isoelectric point. Such a different compound can also be a further protein which is bound to a protein according to the invention, for example via bifunctional chemical compounds. Derivatization also means covalent binding to a macromolecular carrier, or non-covalent inclusion in suitable macromolecular cage structures. Derivatizations can, for example, affect the substrate specificity or the binding strength to the substrate or bring about a temporary blocking of the enzymatic activity if the coupled substance is an inhibitor. This can be useful, for example, for the period of storage. Such modifications may also affect stability or enzymatic activity. Furthermore, they can also serve to reduce the allergenicity and/or immunogenicity of the protein and thus, for example, to increase its skin tolerance. For example, couplings with macromolecular compounds, for example polyethylene glycol, can improve the protein in terms of stability and/or skin tolerance.

In the widest sense, derivatives of a protein according to the invention can also be understood to mean preparations of these proteins. Depending on how it is obtained, processed or prepared, a protein can be combined with various other substances, for example from the culture of the producing microorganisms. A protein can also have other substances added to it, for example to increase its storage stability. All preparations of a protein according to the invention are therefore also in accordance with the invention. This is also independent of whether it actually develops this enzymatic activity in a specific preparation or not. Because it can be desirable that it has little or no activity during storage and only develops its enzymatic function at the time of use. This can be controlled, for example, via corresponding accompanying substances. In particular, the joint preparation of proteases with specific inhibitors is possible in this regard.

Of all the proteases or protease variants and/or derivatives described above, particular preference is given in the context of the present invention to those whose storage stability and/or cleaning performance is improved compared to the starting variant, with the cleaning performance in a washing system being determined as described above.

A further object of the invention is a nucleic acid which codes for a protease according to the invention, and a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.

These can be DNA or RNA molecules. They can be present as a single strand, as a single strand complementary to this single strand, or as a double strand. Particularly in the case of DNA molecules, the sequences of both complementary strands must be taken into account in all three possible reading frames. It should also be taken into account that different codons, ie base triplets, can code for the same amino acids, so that a specific amino acid sequence can be coded by a number of different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences which can encode one of the proteases described above are included in this subject matter of the invention. The person skilled in the art is able to determine these nucleic acid sequences unequivocally since, despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Therefore, starting from an amino acid sequence, the person skilled in the art can easily determine nucleic acids coding for this amino acid sequence. Furthermore, in the case of nucleic acids according to the invention, one or more codons can be replaced by synonymous codons. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Each organism, for example a host cell of a production strain, has a specific codon usage. Codon usage is the translation of the genetic code into amino acids by the organism in question. Bottlenecks in protein biosynthesis can occur if the codons on the nucleic acid in the organism face a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid, this results in a codon being translated less efficiently in the organism than a synonymous codon coding for the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be translated more efficiently in the organism.

A person skilled in the art can use methods that are generally known today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and/or protein chemical methods, to use known DNA and/or amino acid sequences to produce the corresponding nucleic acids up to complete genes to manufacture. Such methods are, for example, from Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press . known.

In the context of the present invention, vectors are understood to mean elements consisting of nucleic acids which contain a nucleic acid according to the invention as a characterizing nucleic acid region. They are able to establish this as a stable genetic element in a species or a cell line over several generations or cell divisions. Vectors are special plasmids, i.e. circular genetic elements, particularly when used in bacteria. In the context of the present invention, a nucleic acid according to the invention is cloned into a vector. The vectors include, for example, those whose origins are bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements from a wide variety of origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the relevant host cells over several generations. They can exist extrachromosomally as separate units or integrate into a chromosome or chromosomal DNA.

Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and there to express a nucleic acid contained therein. Expression is influenced in particular by the promoter or promoters that regulate transcription. In principle, the expression can be effected by the natural promoter originally located in front of the nucleic acid to be expressed, but also by a promoter of the host cell provided on the expression vector or by a modified or a completely different promoter of another organism or another host cell. In the present case, at least one promoter for the expression of a nucleic acid according to the invention is made available and used for its expression. Expression vectors can also be regulated, for example by changing the cultivation conditions or when a certain cell density of the host cells they contain has been reached or by adding certain substances, in particular activators of gene expression. An example of such a substance is the galactose derivative isopropyl-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the nucleic acid contained in cloning vectors is not expressed.

A further object of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention, or which contains a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell. A nucleic acid according to the invention or a vector according to the invention is preferably transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, individual components, ie nucleic acid parts or fragments of a nucleic acid according to the invention can also be introduced into a host cell in such a way that the resulting host cell contains a nucleic acid according to the invention or a vector according to the invention. This procedure is particularly suitable when the host cell already has one or more components of a contains nucleic acid according to the invention or a vector according to the invention and the other components are then supplemented accordingly. Methods for transforming cells are established in the prior art and well known to those skilled in the art. In principle, all cells are suitable as host cells, ie prokaryotic or eukaryotic cells. Such host cells are preferred which can be handled genetically in an advantageous manner, for example with regard to the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handling. This concerns, for example, easy culturing, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenically) expressed protein into the medium surrounding the host cells. Furthermore, the proteases can be modified by the cells producing them after their production, for example by attachment of sugar molecules, formylation, amination, etc. Such post-translational modifications can affect the protease functionally.

Further preferred embodiments are those host cells whose activity can be regulated on the basis of genetic regulatory elements which are made available, for example, on the vector but which can also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions or when a certain cell density is reached, expression can be stimulated. This enables the proteins according to the invention to be produced economically. An example of such a connection is IPTG as described above.

Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on the cultivation conditions. Inexpensive cultivation processes or manufacturing processes can be established as a result. In addition, the specialist in bacteria in fermentation technology has a wealth of experience. Gram-negative or Gram-positive bacteria can be suitable for a special production for a wide variety of reasons, which are to be determined experimentally in the individual case, such as nutrient sources, product formation rate, time requirement, etc.

In gram-negative bacteria such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications. Furthermore, gram-negative bacteria can also be designed in such a way that they smuggle out the expressed proteins not only into the periplasmic space, but also into the medium surrounding the bacterium. In contrast, Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of the Actinomycetales do not have an outer membrane, so that secreted proteins are immediately released into the medium surrounding the bacteria, usually the nutrient medium from which the expressed proteins can be purified. They can be isolated directly from the medium or processed further. In addition, gram-positive bacteria are related or identical to most of the organisms of origin for technically important enzymes and usually form comparable enzymes themselves, so that they have a similar codon usage and their protein synthesis apparatus is naturally aligned accordingly.

Host cells according to the invention can be modified in terms of their requirements for the culture conditions, have other or additional selection markers or express other or additional proteins. In particular, the host cells can also be those which transgenically express a number of proteins or enzymes.

The present invention can in principle be applied to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and results in proteins according to the invention being able to be produced through the use of such microorganisms. Such microorganisms then represent host cells within the meaning of the invention.

In a further embodiment of the invention, the host cell is characterized in that it is a bacterium, preferably one selected from the group of genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum , Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

However, the host cell can also be a eukaryotic cell, which is characterized in that it has a cell nucleus. A further object of the invention is therefore a host cell which is characterized in that it has a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are able to post-translationally modify the formed protein. Examples are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis, which make such systems possible. The modifications that eukaryotic systems carry out, particularly in connection with protein synthesis, include, for example, the binding of low-molecular compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, such as cellulases, can also be advantageous. Furthermore, for example, thermophilic fungal expression systems are particularly suitable for expressing temperature-stable proteins or variants.

The host cells according to the invention are cultivated and fermented in the usual way, for example in discontinuous or continuous systems. In the first case, a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after a period of time to be determined experimentally. Continuous fermentations are characterized by the achievement of a steady state in which cells partially die but also regrow over a comparatively long period of time and at the same time the protein formed can be removed from the medium.

1. a) Kultivieren einer erfindungsgemäßen Wirtszelle, und
2. b) Isolieren der Protease aus dem Kulturmedium oder aus der Wirtszelle.

Host cells according to the invention are preferably used to produce proteases according to the invention. A further subject matter of the invention is therefore a method for the production of a protease

1. a) culturing a host cell according to the invention, and
2. b) isolating the protease from the culture medium or from the host cell.

This subject matter of the invention preferably comprises fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method for the product produced, for example the proteases according to the invention. All fermentation processes that are based on a corresponding process for the production of a protease according to the invention represent embodiments of this subject matter of the invention.

Fermentation processes which are characterized in that the fermentation is carried out using a feed strategy are particularly suitable. Here, the media components that are consumed by the ongoing cultivation are fed. As a result, considerable increases can be achieved both in the cell density and in the cell mass or dry mass and/or in particular in the activity of the protease of interest. Furthermore, the fermentation can also be designed in such a way that unwanted metabolites are filtered out or neutralized by adding buffers or appropriate counterions.

The protease produced can be harvested from the fermentation medium. Such a fermentation process is preferred to isolation of the protease from the host cell, ie product processing from the cell mass (dry matter), but requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems so that the host cells Secrete protease into the fermentation medium. Alternatively, without secretion, the isolation of the Protease from the host cell, ie purification of the same from the cell mass, takes place, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

All of the above issues can be combined into methods to produce the protease of the invention.

Another object of the invention is an agent which is characterized in that it contains a protease according to the invention as described above. The agent is preferably a washing or cleaning agent.

This subject of the invention includes all conceivable types of washing or cleaning agents, both concentrates and agents to be used undiluted, for use on a commercial scale, in the washing machine or for hand washing or cleaning. These include, for example, detergents for textiles, carpets, or natural fibers, for which the term detergent is used. This also includes, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tile, stone, painted surfaces, plastics, wood or leather, for which the term detergent is used, i.e. in addition to manual and machine Dishwashing detergents, for example, also scouring agents, glass cleaners, toilet deodorizers, etc. The detergents and cleaning agents within the scope of the invention also include washing aids that are added to the actual detergent in manual or machine washing in order to achieve a further effect. Furthermore, detergents and cleaning agents within the scope of the invention also include textile pre-treatment and after-treatment agents, i.e. agents with which the item of laundry is brought into contact before the actual wash, for example to loosen stubborn dirt, and also agents that are in one of the the actual textile washing step, give the laundry further desirable properties such as a pleasant feel, crease resistance or low static charge. The latter means include fabric softeners.

The washing or cleaning agents according to the invention, which can be in the form of powdered solids, in post-compacted particle form, as gels, homogeneous solutions or suspensions, can contain, in addition to a protease according to the invention, all known ingredients that are customary in such agents, with preferably at least one further ingredient in the agent is available. The detergents according to the invention can contain, in particular, surfactants, builders (builders), peroxygen compounds or bleach activators. They can also contain water-miscible organic solvents, other enzymes, sequestering agents, electrolytes, pH regulators and/or other auxiliaries such as optical brighteners, graying inhibitors, foam regulators, and dyes and fragrances, and combinations thereof.

In particular, a combination of a protease according to the invention with one or more other ingredient(s) of the agent is advantageous, since such an agent in preferred configurations according to the invention has an improved cleaning performance due to the resulting synergisms. Such a synergism can be achieved in particular by combining a protease according to the invention with a surfactant and/or a builder (builder) and/or a peroxygen compound and/or a bleach activator. However, in preferred embodiments, the agent according to the invention cannot contain any boric acid.

Advantageous ingredients of agents according to the invention are disclosed in the international patent application WO2009/121725A1 , starting there on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. Reference is expressly made to this disclosure and the disclosure content there is included in the present patent application.

An agent according to the invention advantageously contains the protease in an amount of 2 μg to 20 mg, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg and very particularly preferably 50 μg to 10 mg per g of the agent. In various embodiments, the concentration of the protease (active enzyme) described herein in the agent is >0 to 1% by weight, preferably 0.001 to 0.1% by weight, based on the total weight of the agent or composition. Furthermore, the protease contained in the agent and/or other ingredients of the agent can be coated with a substance which is impermeable to the enzyme at room temperature or in the absence of water and which becomes permeable to the enzyme under the conditions in which the agent is used. Such an embodiment of the invention is thus characterized in that the protease is coated with a substance which is impermeable to the protease at room temperature or in the absence of water. Furthermore, the washing or cleaning agent itself can also be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.

1. (a) in fester Form vorliegt, insbesondere als rieselfähiges Pulver mit einem Schüttgewicht von 300 g/l bis 1200 g/l, insbesondere 500 g/l bis 900 g/l, oder
2. (b) in pastöser oder in flüssiger Form vorliegt, und/oder
3. (c) in gelförmiger oder dosierbeutelförmiger (Pouches) Form vorliegt, und/oder
4. (d) als Einkomponentensystem vorliegt, oder
5. (e) in mehrere Komponenten aufgeteilt ist.

In further embodiments of the invention, the agent is characterized in that it

1. (a) is in solid form, in particular as a free-flowing powder with a bulk density of 300 g/l to 1200 g/l, in particular 500 g/l to 900 g/l, or
2. (b) is in paste or liquid form, and/or
3. (c) in gel or pouch form, and/or
4. (d) is present as a one-component system, or
5. (e) is divided into several components.

These embodiments of the present invention include all solid, powdery, liquid, gel-like or pasty dosage forms according to the invention, which can optionally also consist of several phases and can be in compressed or non-compressed form. The agent can be in the form of a free-flowing powder, in particular with a bulk density of 300 g/l to 1200 g/l, in particular 500 g/l to 900 g/l or 600 g/l to 850 g/l. The solid dosage forms of the agent also include extrudates, granules, tablets or pouches. Alternatively, the agent can also be liquid, gel-like or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste. Liquid agents are generally preferred. Furthermore, the agent can be in the form of a one-component system. Such funds consist of one phase. Alternatively, a remedy can also consist of several phases. Such a remedy is therefore divided into several components.

Detergents or cleaning agents according to the invention can contain only one protease. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in a concentration appropriate for the effectiveness of the agent. A further embodiment of the invention is thus represented by agents which also comprise one or more further enzymes. All enzymes which can develop a catalytic activity in the agent according to the invention can preferably be used as further enzymes, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase, Oxidase, oxidoreductase or another protease which can be distinguished from the proteases according to the invention, and mixtures thereof. Further enzymes are advantageously contained in the agent in an amount of 1×10 ^-8 to 5% by weight, based on active protein. Increasingly preferred is each additional enzyme in an amount of from 1×10 ^-7 to 3% by weight, from 0.00001 to 1% by weight, from 0.00005 to 0.5% by weight, from 0.0001 up to 0.1% by weight and particularly preferably from 0.0001 to 0.05% by weight in agents according to the invention, based in each case on active protein. The enzymes particularly preferably show synergistic cleaning performance against certain types of soiling or stains, ie the enzymes contained in the agent composition support each other in their cleaning performance. Such a synergism is very particularly preferred between the protease contained according to the invention and another enzyme of an agent according to the invention, including in particular between said protease and an amylase and/or a lipase and/or a mannanase and/or a cellulase and/or a pectinase . Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the agent according to the invention.

In the cleaning agents described herein, the enzymes to be used can also be formulated together with accompanying substances, for example from the fermentation. In liquid formulations, the enzymes are preferably used as enzyme liquid formulation(s).

As a rule, the enzymes are not provided in the form of the pure protein, but rather in the form of stabilized preparations that can be stored and transported. These ready-made preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, in particular in the case of liquid or gel-like agents, Solutions of the enzymes, advantageously as concentrated as possible, low in water and/or mixed with stabilizers or other auxiliaries.

Alternatively, the enzymes can be encapsulated for both the solid and the liquid dosage form, for example by spray drying or extrusion of the enzyme solution together with a preferably natural polymer, or in the form of capsules, for example those in which the enzymes are enclosed as in a set gel or in those of the core-shell type, in which an enzyme-containing core is coated with a water, air and/or chemical impermeable protective layer. Additional active substances, for example stabilizers, emulsifiers, pigments, bleaching agents or dyes, can also be applied in superimposed layers. Such capsules are applied by methods known per se, for example by shaking or rolling granulation or in fluid-bed processes. Advantageously, such granules, for example due to the application of polymeric film formers, produce little dust and are stable in storage due to the coating.

Furthermore, it is possible to formulate two or more enzymes together, so that a single granulate has several enzyme activities.

The enzymes can also be incorporated into water-soluble films such as are used, for example, in the formulation of detergents and cleaning agents in unit dose form. Such a film allows the enzymes to be released upon contact with water. As used herein, "water soluble" refers to a film structure that is preferably completely water soluble. Such a film preferably consists of (completely or partially hydrolyzed) polyvinyl alcohol (PVA).

Another subject of the invention is a method for cleaning textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one method step, or that a protease according to the invention becomes catalytically active in at least one method step, in particular such that the protease in an amount of 40 μg to 4 g, preferably from 50 μg to 3 g, particularly preferably from 100 μg to 2 g and very particularly preferably from 200 μg to 1 g or in the concentrations described herein.

In various embodiments, the method described above is characterized in that the protease is used at a temperature of from 0 to 100°C, preferably from 0 to 60°C, more preferably from 20 to 40°C and most preferably at 25°C becomes.

This includes both manual and machine methods, with machine methods being preferred. Processes for cleaning textiles are generally characterized by the fact that various active cleaning substances are applied to the The items to be cleaned are applied and washed off after the exposure time, or that the items to be cleaned are treated in some other way with a detergent or a solution or dilution of this agent. The same applies to processes for cleaning all materials other than textiles, especially hard surfaces. All conceivable washing or cleaning methods can be enriched in at least one of the method steps by the use of a washing or cleaning agent according to the invention or a protease according to the invention and then represent embodiments of the present invention. All facts, objects and embodiments for the protease according to the invention and containing it Means are described are also applicable to this subject of the invention. Therefore, at this point, reference is expressly made to the disclosure at the appropriate point, with the indication that this disclosure also applies to the above methods according to the invention.

Since proteases according to the invention naturally already have hydrolytic activity and also develop this in media that otherwise have no cleaning power, such as in mere buffer, an individual and / or the only step of such a method can consist in using a protease according to the invention as the only cleaning-active component is contacted with the soil, preferably in a buffer solution or in water. This represents a further embodiment of this subject matter of the invention.

Alternative embodiments of this subject matter of the invention also represent processes for the treatment of textile raw materials or for textile care, in which a protease according to the invention becomes active in at least one process step. Among these, processes for textile raw materials, fibers or textiles with natural components are preferred, and particularly for those with wool or silk.

Finally, the invention also covers the use of the proteases described herein in detergents or cleaning agents, for example as described above, for the (improved) removal of protein-containing soiling, for example from textiles or hard surfaces. In preferred embodiments of this use, the protease in the washing or cleaning agent is 3 or more days, 4 or more days, 7 or more days, 10 or more days, 12 or more days, 14 or more days, 21 or more days or 28 or more days stored.

All facts, objects and embodiments described for the protease according to the invention and agents containing it are also applicable to this object of the invention. Therefore, at this point, reference is expressly made to the disclosure at the appropriate point, with the indication that this disclosure also applies to the above use according to the invention.

examples Example 1: Overview of the mutations

The invention relates to an alkaline protease of the subtilisin type from Bacillus lentus. From a starting variant (protease according to SEQ ID NO:2 WO2013/060621A1 ) variants were produced by random mutagenesis, which were then screened for improved washing performance and/or enzyme stability, among other things. In this way, mutants with improved storage stability and/or improved cleaning performance were generated from the protease 17 mentioned. variant Amino acid substitutions relative to SEQ ID NO: 1 exit variant S3T V4I R99E V199I mutant 1 S3T V4I R99E V199I Q200L Y203W mutant 2 S3T V4I R99E V199I N212S mutant 3 S3T V4I R99E V199I N74D mutant 4 S3T V4I R99E V199I S154D L256E mutant 5 S3T V4I R99E V199I S154D Q200L Y203W L256E mutant 6 S3T V4I R99E V199I N74D Q200L Y203W mutant 7 S3T V4I R99E V199I N74D S154D Q200L Y203W L256E mutant 8 S3T V4I R99E V199I N74D N212S mutant 9 S3T V4I R99E V199I N74D S154D Y203W L256E mutant 10 S3T V4I R99E V199I N74D Y203W mutant 11 S3T V4I R99E V199I N74D S154D Q200L L256E mutant 12 S3T V4I R99E V199I N74D Q200L mutant 13 S154D Q200L Y203W mutant 14 Q200L Y203W L256E mutant 15 A136Q R143W Y161T Q200L mutant 16 N74D R143Y Y203W N212S L256E mutant 17 A136Q S154D V171L Q200L

Detergent matrix used chemical name % by weight of active substance in the raw material % by weight of active substance in the formulation water demin. 100 rest alkyl benzene sulfonic acid 96 9-18 anionic surfactants 70 3-8 C12-C18 fatty acid Na salt 30 2-4 Non-ionic surfactants 100 5-14 phosphonate 60 0.5-2 citric acid 100 3-5 NaOH 50 0.5-2 defoamer 100 <1% glycerin 99.5 1-3 1,2-propanediol 100 8-12 monoethanolamine 100 4-8 Soil repellent polymer 30 0.5-1 protease stabilizer 100 0.5-1.5 without opt. brighteners, perfume, dye and enzymes. Dosage 3.17 g/L

Example 2: Determination of the storage stability storage

The proteases are present in bioreactor-generated supernatants in Bacillus licheniformis . They are diluted to an equal activity level. 90% detergent matrix without boric acid was mixed with 10% appropriately diluted Bacillus licheniformis supernatant and mixed well. The sealed vessels were stored at 40°C for four weeks. The amount of sample taken was dissolved in 0.1M Tris/HCl (pH 8.6) by stirring for 20 min at room temperature. Thereafter, the AAPF assay was performed as described below.

Protease Activity Assay

The activity of the protease is determined by the release of the chromophore para-nitroaniline from the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachem L-1400). The release of the pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity.

The measurement was carried out at a temperature of 25°C, pH 8.6 and a wavelength of 410nm. The measurement time was 5 minutes with a measurement interval of 20 to 60 seconds.

• 10 µL AAPF-Lösung (70 mg/mL)
• 1000 µL Tris/HCl (0,1 M; pH 8,6 mit 0,1 % Brij 35)
• 10 µL verdünnte Protease-Lösung
• Kinetik erstellt über 5 min bei 25°C (410nm)

measurement approach:

• 10 µL AAPF solution (70 mg/mL)
• 1000 µL Tris/HCl (0.1 M; pH 8.6 with 0.1% Brij 35)
• 10 µL diluted protease solution
• Kinetics generated over 5 min at 25°C (410nm)

The residual activity in % of the residual activity of the starting variant after 4 weeks of storage at 40°C in the above-mentioned detergent matrix is shown below: variant Residual activity (in %) exit variant mutant 1 +29 mutant 2 +9 mutant 5 +60

Variants 1 and 2 show a higher residual activity compared to the starting variant.

Example 3: Determination of cleaning performance mini wash test

• Bedingungen: 40°C, 16° dH Wasser, 1h
• Anschmutzung: CFT CS038

Wash test with Bacillus subtilis culture supernatants containing the screened protease mutants by heterologous expression. The supernatants are used with the same activity as the benchmark = starting variant with a commercially available concentration for proteases in detergents. In contrast to the determination of the storage stability, the samples are not stored, but the cleaning performance is determined directly. The mutants are all related to the washing performance of the starting variant, which is set equal to 100%.

• Conditions: 40°C, 16° dH water, 1h
• Soiling: CFT CS038

Punched-out fabrics (diameter=10 mm) were placed in microtiter plates, washing liquor preheated to 40° C., final concentration 3.17 g/L, lye and enzyme added to the soiling, incubated for 1 h at 40° C. and 600 rpm, then the soiling rinsed several times with clear water, allowed to dry and determined the brightness with a colorimeter. The lighter the fabric becomes, the better the cleaning performance. The L value = brightness is measured here, the higher the brighter. Stated performance in % based on the starting variant corrected by the performance of the detergent without protease. variant Performance in the wash test at 40°C (relative to the performance of the starting variant) exit variant 100% mutant 1 105% mutant 5 109%

Variants 1 and 5 show an increased washing performance compared to the initial variant.

Proteases according to the invention, in particular variants 1 and 5, consequently show not only improved storage stability, but also improved cleaning performance.

Claims (13)

1. A protease comprising an amino acid sequence which has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 over the total length thereof and which has, based on the numbering according to SEQ ID NO:1, at

   i) the positions corresponding to positions 3, 4, 99 and 199 the amino acid substitutions S3T, V4I, R99E and V199I, and

   ii) at least one of the positions corresponding to positions 200, 203, 209 or 212 at least one amino acid substitution.
2. The protease according to claim 1, wherein the at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212 is selected from the group consisting of 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212N, 2125 and 212T.
3. The protease according to any one of claims 1 to 2, wherein the protease, in each case based on numbering according to SEQ ID NO:1, comprises:

   (i) the amino acid substitutions S3T + V4I + R99E + V199I; and

   (ii) any of the following amino acid substitution combinations: (i) Q200L + Y203W; (ii) N212S; (iii) Q200L + Y203W + S154D + L256E; (iv) N74D + Q200L + Y203W; (v) N74D + S154D + Q200L + Y203W + L256E; (vi) N74D + N212S; (vii) N74D + S154D + Y203W + L256E; (viii) N74D + Y203W; (ix) N74D + S154D + Q200L + L256E; (x) N74D + Q200L; (xi) S154D + Q200L + Y203W; (xii) Q200L + Y203W + L256E; (xiii) A136Q + R143W + Y161T + Q200L; (xiv) N74D + R143Y + A209W + N212S + L256E; (xv) A136Q + S154D + V171L + Q200L.
4. A protease, characterized in that

   (a) it is obtainable from a protease according to any one of claims 1 to 3 as a starting molecule by one or more conservative amino acid substitutions, wherein the protease comprises:

   (i) amino acid substitutions S3T, V4I, R99E and V199I at the positions corresponding to positions 3, 4, 99 or 199; and

   (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212; or

   (b) it is obtainable from a protease according to any one of claims 1 to 3 as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence extending over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids matching the parent molecule, wherein the protease comprises:

   (i) amino acid substitutions S3T, V4I, R99E and V199I at the positions corresponding to positions 3, 4, 99 or 199; and

   (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212.
5. A method for the preparation of a protease, which comprises introducing

   i) amino acid substitutions S3T, V4I, R99E and V199I at the positions corresponding to positions 3, 4, 99 or 199 with respect to the numbering according to SEQ ID NO:1, and

   ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212 with respect to the numbering according to SEQ ID NO:1,

   into a starting molecule having an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 over the total length thereof.
6. The method of claim 5, further comprising one or more of the following method steps:

   (a) introducing a single or multiple conservative amino acid substitution, wherein the protease comprises:

   (i) amino acid substitutions S3T, V4I, R99E and V199I at the positions corresponding to positions 3, 4, 99 or 199; and

   (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209, or 212;

   (b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence that is at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids matching the parent molecule, wherein the protease has:

   (i) amino acid substitutions S3T, V4I, R99E and V199I at the positions corresponding to positions 3, 4, 99 or 199; and

   (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 200, 203, 209 or 212.
7. Nucleic acid encoding a protease according to any one of claims 1 to 4 or encoding a protease obtained by a method according to any one of claims 5 to 6.
8. Vector containing a nucleic acid according to claim 7, in particular a cloning vector or an expression vector.
9. A non-human host cell comprising a nucleic acid according to claim 7 or a vector according to claim 8, or comprising a protease according to any one of claims 1 to 4, or comprising a protease obtained by a method according to any one of claims 5 to 6, in particular one that secretes the protease into the medium surrounding the host cell.
10. A method for the production of a protease comprising

    a) culturing a host cell according to claim 9; and

    b) isolating the protease from the culture medium or from the host cell.
11. Composition, in particular a washing or cleaning composition, characterized in that it comprises at least one protease according to any one of claims 1 to 4 or a protease obtained by a method according to any one of claims 5 to 6.
12. A method for cleaning textiles or hard surfaces, characterized in that a composition according to claim 11 is used in at least one method step.
13. Use of a protease according to any one of claims 1 to 4 or a protease obtained by a method of any one of claims 5 to 6 in a washing or cleaning composition for removing peptide- or protein-containing soiling.